The invention relates to 22S-hydroxycholesta-8,14-diene derivatives, to pharmaceutical compositions containing the same, as well as to the use of these 22S-hydroxycholesta-8,14-diene derivatives for the preparation of a medicament for the control of fertility.
Sexual reproduction involves a cyclic alternation of diploid and haploid states: diploid cells divide by the process of meiosis to form haploid cells, and the haploid cells fuse in pairs at fertilization to form new diploid cells. The process of meiosis is characterized by two meiotic divisions, unique to both male and female germ cells. During the process two cell divisions, following one round of DNA replication, give rise to four haploid cells from one single diploid cell. Chromosomal crossover events, during which paternal and maternal genetic material is exchanged, occur during the prophase of the first meiotic division. At the end of the first meiotic division one member of each chromosome pair, composed of two sister chromatids is distributed to each daughter cell. The second meiotic division segregates each sister chromatide into a separate haploid cell. Male and female germ cells are subject to similar meiotic divisions but differ in the regulation of these processes. In the male meiosis is a continuous process in germ cells derived from a population of immature germ cells, the stem cell spermatogonia. After sexual maturation of the male, spermatogonia from this stem cell population embark on meiosis. The first and second meiotic division proceed without interruption and eventually give rise to four mature spermatozoa.
In the female, primary oocytes start the first meiotic division already during the embryonic stage but they remain arrested in the prophase (dictyate stage) until the female becomes sexually mature. Meiosis resumes at the time of ovulation (egg maturation) after which the first meiotic division is completed and the second meiotic division is initiated. In most vertebrates the second meiotic division is arrested at the metaphase and only completed after fertilization. At the end of the first and of the second meiotic division the cytoplasm divides asymmetrically to produce two secondary oocytes, each with a haploid number of single chromosomes, but greatly differing in size: one is a small polar body, which eventually degenerates, and the other is a large cell containing all the developmental potential. Finally one mature ovum is produced.
The stage at which the developing oocyte is released from the ovary and is ready for fertilization differs in different species. In both invertebrates and vertebrates ovarian accessory cells respond to polypeptides (gonadotropins) produced elsewhere in the body so as to control the maturation of the oocyte and eventually (in most species) ovulation. In humans the primary oocytes of the newborn female are arrested in prophase of meiotic division I and most are surrounded by a single layer of follicle cells; such an oocyte with its surrounding cells constitute the primordial follicle. A small portion of primordial follicles sequentially begins to grow to become developing follicles: the follicle cells enlarge and proliferate to form a multilayered envelope around the primary oocyte; the oocyte itself enlarges and develops the zona pellucida, an extracellular matrix consisting largely of glycoproteins, and cortical granules, specialized secretory vesicles just under the plasma membrane in the outer region, the cortex, of the egg cytoplasm [when the egg is activated by a sperm, these cortical granules release their contents by exocytosis; the contents of the granules act to alter the egg coat so as to prevent other sperms from fusing with the egg].
The developing follicles grow continuously and some of them develop a fluid-filled cavity, or antrum, to become antral follicles. Development of such follicles is dependent on gonadotropins (mainly follicle stimulating hormone -FSH) secreted by the pituitary gland and on estrogens secreted by the follicle cells themselves. Starting at puberty, a surge of secretion by the pituitary of another gonadotropin, luteinizing hormone (LH), activates a single antral follicle to complete its development: the enclosed primary oocyte matures to complete the meiotic division I as the stimulated follicle rapidly enlarges and ruptures at the surface of the ovary, releasing the secondary oocyte within. As is the case with most mammals, the secondary oocyte is triggered to undergo division II of meiosis only if it is fertilized by a sperm.
Studies on the mechanisms controlling initiation and regulation of the meiotic process in male and female germ cells suggest a role for cyclic nucleotides in mediating meiotic arrest. Spontaneous maturation of oocytes can be prevented by compounds that maintain elevated cAMP levels [Eppig, J. and Downs, S. (1984) Biol. Reprod. 30: 1-11]. Purines, like adenosine or hypoxanthine, are thought to be involved in the cAMP mediated maintenance of meiotic arrest [Eppig, J., Ward-Bailey, P. and Coleman, D. (1985) Biol. Reprod. 33: 1041-1049]. The presence of a meiosis regulating substance in a culture system of fetal mouse gonads was first described by Byskov, A. et al (1976) Dev. Biol. 52: 193-200. It was suggested that the concentrations of a meiosis activating substance (MAS) and a meiosis preventing substance (MPS) regulate the meiotic process in concert [Byskov, A. et al. (1994). In xe2x80x9cThe physiology of reproductionxe2x80x9d, Eds. Knobil, E. and Neill, J., Raven Press, New York]. More recently (3xcex2,5xcex1,20R)-4,4-dimethylcholesta-8,14,24-trien-3-ol (FF-MAS), isolated from human follicular fluid, and (3xcex2,5xcex1,20R)-4,4-dimethylcholesta-8,24-dien-3-ol, isolated from bull testes, were identified by Byskov, A. et al [(1995), Nature 374: 559-562] as endogenous meiosis activating substances in human and bovine, respectively. These sterols proved to be able to activate the resumption of meiosis in cultured cumulus enclosed and naked mouse oocytes.
Derivatives of the endogenous sterols, having either a saturated or an unsaturated cholestane side chain, have been disclosed in the international patent applications WO 96/00235, WO97/00884 and WO98/28323 (NOVO NORDISK A/S) as meiosis regulating substances. Meiosis regulating substances are compounds that are agonists or antagonists of a naturally occurring meiosis activating substance. Thus, they might be used in the treatment of infertility or for contraception.
A drawback of the compounds described in the first two patent applications mentioned above is their restricted membrane penetration, which results in accumulation in cell membranes or fatty tissues, thereby restricting their therapeutic potential as fertility control agents. This unwanted accumulation is most probably due to the fysico-chemical properties of these compounds, which resemble those of cholesterol, an important constituent of cell membranes [Norum, K. R. et al (1983) Physiological Rev. 63: 1343; Babiker, A. et al (1998) Biochimica and Biophysica Acta 1392: 333].
We have now found, that certain 22S-hydroxy substituted cholestane derivatives, i.e. the 22S-hydroxycholesta-8,14-diene derivatives of formula I given below, are highly active as meiosis activating compounds. 
wherein
R1 is OR, OSO3H or xe2x95x90NOR; with R being H, (C1-6)alkyl or (C1-6)acyl;
each of R2 and R3 is independently hydrogen or (C1-6)alkyl;
R4 is hydrogen, (C1-6)alkyl or (C1-6)acyl;
R5 is hydrogen; or R5 designates, together with R6, an additional bond between the carbon atoms at which R5 and R6 are placed;
R6 is hydrogen, hydroxy or halogen; or R6 designates, together with R5, an additional bond between the carbon atoms at which R6 and R5 are placed;
each of R7 and R8 is independently hydrogen or (C1-4)alkyl, optionally substituted with OH, (C1-4)alkoxy, or halogen;
or a pharmaceutically acceptable salt thereof.
An advantage of 22-hydroxycholestanes is their ability to pass cell membranes which would circumvent the problems described above, see Stocco, D. M. (1997), Endocrine 6: 99-109.
The above 22S-hydroxycholesta-8,14-diene derivatives having the general formula I are highly active as meiosis activating substances. In fact, they are active in the CEO assay (see below) wherein even the natural meiosis activating substance FF-Mas is not. This does not follow at all from the prior art. In this respect it is noted that, incidentally, a certain 22R-hydroxy substituted cholestane derivative (22R-hydroxycholesterol) has been disclosed in WO 98/28323, but is an antagonist.
The invention further provides a pharmaceutical composition comprising a 22S-hydroxycholesta-8,14-diene derivative having the general formula I. A further aspect of the invention resides in the use of a 22S-hydroxycholesta-8,14-diene derivative having the general formula I for the manufacture of a medicament for the control of fertility.
The term (C1-6)alkyl as used in the definition of formula I means a branched or unbranched alkyl group having 1-6 carbon atoms, like hexyl, pentyl, butyl, isobutyl, tertiary butyl, propyl, isopropyl, ethyl and methyl. Likewise, the term (C1-4)alkyl means an alkyl group having 1-4 carbon atoms.
The term (C1-6)acyl means an acyl group derived from a carboxylic acid having from 1-6 carbon atoms, like hexanoyl, pentanoyl, pivaloyl, butyryl, propanoyl, acetyl and formyl. Also included within the definition of (C1-6)acyl are acyl groups derived from dicarboxylic acids, like hemi-glutaroyl, hemi-succinoyl, and hemi-maloyl. A preferred (C1-4)acyl group is hemi-succinoyl.
The term (C1-4)alkoxy means an alkyloxy having 1-4 carbon atoms, like butyloxy, propyloxy, isopropyloxy, ethyloxy, and, preferably, methyloxy.
The term halogen means F, Cl, Br or I. Cl and F are preferred, F being most preferred.
It is understood that the 22S-hydroxycholesta-8,14-diene derivatives of the invention have the natural configurations 5xcex1, 10xcex2, 13xcex2, and 17xcex2. The configuration at position 20 of the 22S-hydroxycholesta-8,14-diene derivatives of the invention can be either R or S. Preferred compounds are those with the 20S configuration.
Even more preferred compounds according to the invention are the 22S-hydroxycholesta-8,14-diene derivatives of formula I wherein R1 is OR wherein R has the previously given meaning. Among these preferred compounds those with the 3-OR substituent in the xcex2-configuration are especially preferred. A specifically preferred agonistic compound of the invention is the 22S-hydroxycholesta-8,14-diene derivative (3xcex2,5xcex1,20S,22S)-4,4-dimethylcholesta-8,14,24-triene-3,22-diol.
The 22S-hydroxycholesta-8,14-diene derivatives of this invention have the natural configurations 5xcex1, 10xcex2, 13xcex2, 17xcex2, and possess also one or more additional chiral carbon atoms. The compounds may therefore be obtained as a pure diastereomer, or as a mixture of diastereomers. Methods for obtaining the pure diastereomers are well known in the art, e.g. crystallization or chromatography.
The meiosis activating activity of the 22S-hydroxycholesta-8,14-diene derivatives of the invention is measured in an in vitro oocyte assay as the ability to overcome the hypoxanthine maintained meiotic arrest in denuded oocytes (DO) or cumulus enclosed oocytes (CEO).
The compounds can be used to stimulate meiosis in both male and female and thus can be used as fertility regulating agents. Fertility regulation comprises contraception and infertility treatment.
For female contraception a 22S-hydroxycholesta-8,14-diene derivative according to formula I can be used for induction of premature maturation of oocytes which are still inside the ovary, before the naturally occurring gonadotropin surge [reduced fertility by inducing premature maturation of oocytes has been demonstrated in rats by Mattheij, J. et al (1993), Gynecolo Obstet. Invest. 36: 129-135]. On in vivo administration the compounds of the invention specifically affect germ cells and therefore have the advantage of maintenance of endogenous hormonal levels and subsequently maintenance of normal cycle length. Such a contraceptive method will not cause unwanted side-effects sometimes associated with steroidal contraception (e.g. thrombosis, mood, unscheduled bleeding, malignant breast disease).
A further advantage of the 22S-hydroxycholesta-8,14-diene derivatives of the invention is their inability to induce maturation in incompetent oocytes (isolated from pre-antral follicles), which indicates that the compounds will not affect the entire oocyte reserve in the ovaries. Only oocytes from antral follicles (competent oocytes) can be induced to mature by the compounds of the invention.
For treatment of female infertility caused by the absence of mature oocytes the compounds of the invention can be administered in vivo to timely stimulate the maturation of competent oocytes.
The compounds of the invention can also be used for suppletion of culture media for in vitro fertilization procedures in order to improve oocyte quality.
For treatment of male infertility caused by a shortage of the number of mature spermatozoa the compounds of the invention can be administered in vivo to stimulate the maturation of spermatogonia.
For therapeutic use, salts of the compounds of formula I are those wherein the counterion is pharmaceutically acceptable. However, salts of the acids according to formula I [i.e. compounds wherein R1 is OSO3H] may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound. All salts, whether pharmaceutically acceptable or not, are included within the ambit of the present invention. Examples of salts of acids according to the invention are mineral salts such as sodium salt, potassium salt, and salts derived from organic bases like ammonia, imidazole, ethylenediamine, triethylamine and the like.
The compounds of formula I or a pharmaceutically acceptable salt thereof, also referred to herein as the active ingredient, may be administered enterally or parenterally. The exact dose and regimen of administration of the active ingredient, or a pharmaceutical composition thereof, will necessarily be dependent upon the therapeutic effect to be achieved (contraception or infertility), and will vary with the particular compound, the route of administration, and the age and condition of the individual subject to whom the medicament is to be administered.
In general parenteral administration requires lower dosages than other methods of administration which are more dependent upon adsorption. However, a dosage for humans preferably contains 0.0001-25 mg per kg body weight. The desired dose may be presented as one dose or as multiple subdoses administered at appropriate intervals throughout the day, or, in case of female recipients, as doses to be administered at appropriate daily intervals throughout the menstrual cycle. The dosage as well as the regimen of administration may differ between a female and a male recipient.
In case of in vitro or ex vivo applications, the compounds of the inventions are to be used in the incubation media in a concentration of approximately 0.01-5 xcexcg/ml.
The present invention thus also relates to pharmaceutical compositions comprising a 22S-hydroxycholesta-8,14-diene derivative according to formula I in admixture with pharmaceutically acceptable auxiliaries, and optionally other therapeutic agents. The auxilliaries must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipients thereof.
Pharmaceutical compositions include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The compositions may be prepared by any method well known in the art of pharmacy, for example, using methods such as those described in Gennaro et al., Remington""s Pharmaceutical Sciences (18th ed., Mack Publishing Company, 1990, see especially Part 8: Pharmaceutical Preparations and Their Manufacture). Such methods include the step of bringing in association the active ingredient with any auxilliary agent. The auxilliary agent(s), also named accessory ingredients, include those conventional in the art (Gennaro, supra), such as, fillers, binders, diluents, disintegrants, lubricants, colorants, flavoring agents and wetting agents.
Pharmaceutical compositions suitable for oral administration may be presented as discrete dosage units such as pills, tablets or capsules, or as a powder or granules, or as a solution or suspension. The active ingredient may also be presented as a bolus or paste. The compositions can further be processed into a suppository or enema for rectal administration.
For parenteral administration, suitable compositions include aqueous and non-aqueous sterile injection. The compositions may be presented in unit-dose or multi-dose containers, for example sealed vials and ampoules, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of sterile liquid carrier, for example, water prior to use.
Compositions, or formulations, suitable for administration by nasal inhalation include fine dusts or mists which may be generated by means of metered dose pressurised aerosols, nebulisers or insufflators.
The 22S-hydroxycholesta-8,14-diene derivatives of the invention can also be administered in the form of implantable pharmaceutical devices, consisting of a core of active material, encased by a release rate-regulating membrane. Such implants are to be applied subcutaneously or locally, and will release the active ingredient at an approximately constant rate over relatively large periods of time, for instance from weeks to years. Methods for the preparation of implantable pharmaceutical devices as such are known in the art, for example as described in European Patent 0,303,306.
The compounds of the invention may be produced by various methods known in the art of organic chemistry in general, and especially in the art of the chemistry of steroids (see, for example: Fried, J. and Edwards, J. A., xe2x80x9cOrganic Reactions in Steroid Chemistryxe2x80x9d, Volumes I and II , Van Nostrand Reinhold Company, New York, 1972). A convenient starting material for the preparation of compounds of formula I is a compound of general formula II , 
wherein R2 and R3 are independently hydrogen or (C1-6)alkyl, R9 is a hydroxy-protecting group such as an acyl group, like an acetyl group, a benzoyl group or a pivaloyl group, or an alkoxyalkyl group, like an ethoxyethyl group or a tetrahydropyranyl (THP) group, whose preparation is described in WO-09852965 and WO-09855498. Suitable protective groups are known in the art [for example from Greene, T. W. and Wuts, P. G. M.: Protective Groups in Organic Synthesis, Second Edition, Wiley, N.Y., 1991.
Starting from this key intermediate, the side-chain is constructed, using methods known in the art [see: Redpath, J. et al, Chem. Soc. Rev. 12, 75 (1983); Zhu, G.-D. et al, Chem. Rev. 95, 1877 (1995); Apfel, M. A., J. Org. Chem. 44, 643 (1979); Kircher, H. W. et al, J. Org. Chem. 52, 2586 (1987); Takeshita, T. et al, Chem. Pharm. Bull. 24, 1928 (1976); Dasgupta, S. K. et al, J. Org. Chem. 39, 1658 (1974); Dolle, R. E. et al, J. Am. Chem. Soc. 111, 278 (1989); Poyser, J. P. et al, J. Chem. Soc., Perkin Trans. I, 2061 (1974)].
For instance, compounds of formula I (R1xe2x95x90OH, R4xe2x95x90H, R5xe2x95x90H) can be prepared by reaction of compounds of formula II with a suitably substituted alkylmetallic reagent of formula III, 
wherein M is Li, MgX, ZnX, or CeX (Xxe2x95x90Cl,Br,I), R7 and R8 have the previously given meaning, any hydroxy group present in R7 or R8 being suitably protected, and R10 is H, protected OH, or halogen, which often results in the predominant formation of the 22S-hydroxycholestane derivative [see e.g. Poyser, J. P. et al, J. Chem. Soc., Perkin Trans. I, 2061 (1974)]. The 22S-hydroxy- and 22R-hydroxy derivatives can be separated or, if necessary, the 22R-hydroxy isomer is epimerized to the 22S-hydroxy compound. Epimerization at C-22 can be accomplished e.g. by means of a Mitsunobu reaction [see Hughes, D. L., Organic Reactions 42, 335 (1992)], or by treatment with methanesulfonyl chloride or p-toluenesulfonyl chloride followed by reaction with an oxygen nucleophile [e.g. potassium superoxide, see Corey, E. J. et al, Tetrahedron Lett. 3183 (1975) and Larock, R. C., xe2x80x9cComprehensive Organic Transformationsxe2x80x9d, VCH Publishers, Inc.,1989, p. 479]. In both cases, removal of any remaining protective groups then results in compounds of formula I (R1xe2x95x90OH; R4,R5xe2x95x90H).
Compounds of formula I (R1xe2x95x90OH, R4xe2x95x90H, R5 and R6 together form an additional bond, i.e. a xcex9424 double bond) can be obtained from aldehyde II as follows: the latter is reacted with the anion of acetonitrile [MCH2Cxe2x89xa1N, Mxe2x95x90Li, Na, K, MgX, ZnX; see: Arseniyadis, S. et al, Org. React. 31, 1 (1984)] to give a diastereomeric pair of 22R and 22S-hydroxycholane-24-nitrile derivatives. After isolation of the 22S-hydroxy epimer the 22-hydroxy group is protected as a silyl ether or as an alkoxyalkyl ether. If necessary, the 3-hydroxy group is reprotected in the same way, or with an orthogonal protective group. The cyano group is reduced to the corresponding carboxaldehyde group by treatment with a reducing agent such as diisobutylaluminium hydride or other reducing agents capable of converting a carbonitrile group into a carboxaldehyde group. Wittig reaction with a suitably substituted Wittig reagent and removal of protective groups then results in the cholest-24-enes of formula I (R1xe2x95x90OH, R4xe2x95x90H, R5 and R6 together form a xcex9424 double bond).
For methods used for the Wittig olefination reaction, see Maercker, A., Org. React. 14, 270 (1965). Alternatively, Peterson reactions can be used, see Ager, D. J. Org. React. 38, 1 (1990).
The Wittig reaction can also be performed in the opposite direction. In that case, the cholan-24-al derivative mentioned above is reduced to the corresponding cholan-24-ol derivative with the use of reducing agents like for example lithium aluminium hydride, sodium borohydride, or other hydride reducing agents known in the art. The 24-hydroxy group is converted to a leaving group, e.g. Br, I, mesyloxy, or tosyloxy. Reaction with an appropriate phosphine (e.g. triphenyl phosphine), Wittig reaction with a suitably substituted ketone, and finally, removal of protective groups then results in the formation of compounds of formula I (R1xe2x95x90OH, R4xe2x95x90H, R5 and R6 together form a xcex9424 double bond).
Construction of xcex9424-cholestanes I from aldehydes II can also be accomplished by an analogous reaction sequence which makes use of anions of acetic acid or anions of acetic acid esters [see: Petragnani, N. et al, Synthesis, 521 (1982)]. Techniques for homologation are known in the art, see for example Mathieu, J. et al: Formation of Cxe2x80x94C Bonds, Vol. I-III, Georg Thieme Publishers, Stuttgart, 1973.
Selective deprotection of 3-OH and 22-OH enables independent conversion, using methods known in the art, of 3-OH into 3-OR, OSO3H or xe2x95x90NOR (R as previously defined), and of 22-OH into 22-OR4 (R4 as previously defined), respectively.